Mutations in the epidermal growth factor receptor (EGFR) gene in triple negative breast cancer: possible implications for targeted therapy

Abstract

Introduction: Triple negative breast cancer is associated with poorer prognosis and unresponsiveness to endocrine and anti-HER2 directed agents. Despite emerging data supporting the use of polyADP-ribose polymerase (PARP) inhibitors, complete and durable responses are rare and exploration of additional targeted therapies is needed. Epidermal growth factor receptor (EGFR) is expressed in triple negative breast cancer and several clinical trials are testing the role of anti-EGFR directed therapy. However, the rate of EGFR mutations is poorly defined. We, therefore, sought to characterize EGFR mutations in triple negative breast cancers.

Methods: Seventy samples were randomly chosen from a cohort of 653 triple negative breast tumours for EGFR mutation analysis. These samples were immunostained for EGFR protein expression and consisted of negatively stained and positively stained cases. DNA was extracted from paraffin blocks and polymerase chain reaction was performed to amplify exon regions 18 to 21 of the EGFR gene. Direct sequencing of the purified PCR products was performed.

Results: EGFR mutations were found in 8 of 70 samples (11.4%). Mutations were predominantly exon 19 deletions (4 of 70 samples, 5.7%), which clustered in the region spanning codons 746 to 759 within the kinase domain of EGFR. Two types of exon 19 deletions were seen: a 15 nucleotide deletion (del E746-A750) (2 of 70 samples) and a 24 nucleotide deletion (del S752 - I759) (2 of 70 samples). Other exon 19 mutations observed were the inversion of the complementary strand (1 of 70 samples). Exon 21 mutations included missense substitution, L858R (1 of 70 samples) and T847I (2 of 70 samples). Mutations observed were independent of EGFR protein expression determined by immunohistochemical staining.

Conclusions: This study is among the first to document the presence and estimate the prevalence of EGFR mutations in triple negative breast cancer. These findings have potential implications for the design of clinical trials involving anti-EGFR directed therapy which currently do not select for patients based on presence of activating EGFR mutations, which may hence be underpowered to detect significant benefit in unselected populations. More complete sampling of EGFR mutation status in triple negative breast cancer is needed to determine the true mutation rate.

Figure 1

Immunohistochemical staining of EGFR in triple negative breast cancers. (A) Negative EGFR expression (B) 1+ EGFR expression (C) 2+ EGFR expression (D) 3+ EGFR expression.

Figure 2

Exon 19 deletions encountered in triple negative breast cancers. Diagrams A to D show nucleotide sequences of EGFR gene in triple negative breast tumour specimens with heterozygous in-frame deletions within Exon 19 tyrosine kinase domain (double peaks). Tracings in both (A, C) sense and (B, D) antisense directions. The wildtype sequence is shown in capital letters, while the deleted mutant sequence is in lowercase letters. Deleted sequence is highlighted in red capital letters. (A, B) 24 bp deleted region of EGFR gene leading to removal of SPKANKEI at codons 752 to 759. (C, D) 15 bp deleted region of EGFR resulting in deletion of ELREA at codons 746 to 750.

Figure 3

Exchange of positions of double strands in EGFR exon 19, accompanied by gene inversions. Diagrams A and B show nucleotide sequences of EGFR exon 19 gene in triple negative breast tumour specimens with heterozygous gene inversions of the complementary strand (double peaks). Tracings in both (A) sense and (B) antisense directions. The wild type sequence is shown in black capital letters, while the mutant sequence is in red lowercase letters. The complementary sequence of the mutant strand corresponds exactly to the wild type sequence and the orientation is reversed. Note only a segment of the sequencing diagrams (reading sequence towards the end) of the forward and reverse sequence is shown to demonstrate that the whole portion of exon 19 gene is inverted with exchange of the complementary strands.

Figure 4

Exon 21 missense mutations seen in triple negative breast cancers. (A, B) Diagrams show substitution of T to G at mRNA coding nucleotide sequence 2573; leucine to arginine amino acid change at codon 858 (L858R). (C, D) Substitution of C to T at mRNA coding nucleotide sequence 2540, resulting in a threonine to isoleucin substitution at amino acid codon position 847. Tracings in both (A, C) sense and (B, D) antisense directions. The wild type sequence is shown in capital letters, while the missense nucleotide is highlighted in red lowercase letters.